The automatization of nucleic acid sequencing and separation of polynucleotides from sequencing reaction has enabled the rapid and large scale sequencing of a variety of nucleic acids of interest. Typically, nucleic acid sequencing is performed by incorporating chain terminating nucleotides which are labeled with fluorophores or dyes into the sequencing reaction. The different sized nucleic acid products are separated by gel electrophoresis and detected in automated sequencers. Ongoing large scale nucleic acid sequencing projects require that the nucleic acid sequencing process be as efficient as possible. However, existing nucleic acid sequencing methods are limited by the electrophoresis step.
Current methods fail to maximize the amount of useful data generated from sequenced polynucleotides that are separated and detected by automated electrophoresis. With capillary gel electrophoresis it typically takes between 15 and 40 minutes for the first nucleic acid fragments within a sample to be separated and produce useful data in the form of peaks. This initial time where no useful data is produced is known as “sample dead time”, the length of which varies according to the size of the nucleic acid fragments and upon the conditions in which the electrophoresis is performed.
Another factor that limits the rate of useful data produced by current automated capillary gel electrophoresis-based polynucleotide sequencing procedures is that gel is flushed and replaced with a new gel after each sample has been separated and identified by the electrophoresis. This flushing step takes approximately 10 minutes for a complete cycle on a typical automated capillary gel electrophoresis system. Thus, for an electrophoresis run that takes 35 to 150 minutes to perform, useful data is only collected during about 10 to 100 minutes of that electrophoresis run.
What is needed is a electrophoresis method to reduce the sample dead time and increase the efficiency of electrophoresis of nucleic acid samples to enable large scale nucleic acid sequencing projects to be performed more quickly and at an overall lower cost.